Fig. 1.
High-level production of mRFP in C. albicans. (A) Yeast colony color turns from cream to pink when C. albicans (Ca) overproduces yEmRFP. CAI4 was transformed with pADH-yEmRFP and subjected to multiple rounds of selective and then nonselective growth. Note the colony color phenotype of this strain (strain SKY40) and one that contains a single integrated copy of PADH1-yEmRFP (strain SKY52) after growth on SD (−Ura) medium for 2 days at 30°C. An S. cerevisiae (Sc) strain expressing hundreds of copies of yEmRFP on 2μm plasmids is shown for comparison. (B) Fluorescence microscopy of C. albicans expressing multiple copies of yEmRFP. After multiple rounds of selective growth, almost all of the cells in the population (98 to 99%) remain brightly fluorescent after >10 generations on nonselective YPAD. (C) Elevated yEmRFP production in C. albicans containing multiple, stable copies of yEmRFP. Protein extracts were prepared from C. albicans strains with a single integrated copy of PADH1-FLAG-yEmRFP (strain SKY42) or multiple stable copies (strain SKY41), fractionated by 12% SDS-PAGE, and analyzed by Western blotting with anti-FLAG antibodies.